芒果类黄酮糖基转移酶互作蛋白的筛选及其功能分析

Mango fruit coloring was a complex process, mostly concentrated chlorophyll degradation, the anthocyanin biosynthesis genes and regulatory factors of the study in the peel. The content and composition of anthocyanin were to decide the color of the mango fruit the main factors. The UFGT enzyme may catalyze unstable anthocyanidin into anthocyanin, was the last enzyme in the process of synthesis of anthocyanin. It was found that the UFGT gene expression was lower in the green peel, high expression levels in the red peel by semi-quantitative analysis in 'Guifei' Mango. In this study, the full-length cDNA sequences of UFGT was cloned using molecular biology method. The screening its interacting protein, gene fragment as bait, using the yeast two-hybrid. Interaction gene sequence was obtained according to the screening. The full length cDNA sequence was cloned by the method of RACE, and verify UFGT and screening gene interactions using the yeast two-hybrid. The UFGT and screening genes were constructed by gene transfer vector, observation and analysis of the physiological and biochemical phenotype of transgenic plants. It was focuses on the impact of mango fruit anthocyanin synthesis, closely related UFGT of genes and their interaction gene peel coloring, in order to reveal the molecular mechanism of anthocyanin biosynthesis in mango fruit. It was expected that theoretical basis was provided for the regulation and control of the mango fruit coloring in production.

芒果果实的着色是一个比较复杂的过程，多集中于叶绿素的降解、花色素苷的合成相关基因及其调控因子等方面的研究，而花色素苷的含量和组成则是决定红色芒果果实颜色的主要因素。UFGT酶可以把不稳定的花色素催化成花色素苷，是花色素苷合成过程中的最后一个酶。通过对不同时期的'贵妃'芒果半定量PCR分析发现，UFGT基因在绿色果皮中表达较低，而在红色果皮中表达量较高。本研究采用UFGT基因全的片段为诱饵，利用酵母双杂交技术筛选与其相互作用的蛋白。根据筛选得到的互作基因的序列，采用RACE的方法克隆其全长的cDNA序列， 并通过体外和体内实验验证UFGT和筛选蛋白的相互作用。将UFGT和筛选的互作基因分别构建转基因载体，对转基因植物的表型及生理生化进行观测和分析。重点探讨影响红色芒果果实花色素苷合成密切相关的UFGT基因及其互作基因对果皮着色的影响，借以深入揭示芒果果实花色素苷生物合成的分子机理。

芒果果实的着色是一个比较复杂的过程，大多集中于果皮叶绿素的降解、花色素苷的合成相关基因及其调控因子等方面的研究，而花色素苷的含量和组成则是决定红色芒果果实颜色的主要因素。UFGT酶可以把不稳定的花色素催化成花色素苷，是花色素苷合成过程中的最后一个酶。主要研究内容是克隆UFGT基因全长序列，并对该目标基因全长进行序列分析和同源性比较，对蛋白质结构进行预测。以UFGT基因为诱饵进行了酵母双杂交的互作蛋白的筛选，得到相关互作蛋白的序列完成相关蛋白的全长cDNA的克隆，进行转基因番茄的研究，得到相关的表型和生理指标检测结果。本研究从芒果果实中克隆得到了UFGT基因的全长cDNA和基因组序列，该基因开放阅读框为1392 bp，编码463个氨基酸，基因组扩增得到了2770bp长度的片段分析发现，该基因含有2个内含子，分别在501-1095bp，1548-2330bp。通过系统发育分析发现该基因编码的蛋白与荔枝、山竹子等热带果树具有较近的亲缘关系。对UFGT基因的表达进行分析发现：红色的贵妃品种中表达量较高，而黄色的金煌品种中表达量较低。通过酵母双杂交技术筛选了与UFGT蛋白互作的蛋白，克隆相关基因的全长的cDNA序列，共得到13个基因的全长序列及部分基因的基因组DNA的序列。分析了不同色泽芒果果实中花色素苷含量及种类、叶绿素含量、类胡萝卜素等生理指标的变化，表明不同色泽的果实花色素苷和叶绿素含量有一定的差异，分析发现红色和黄色品种中主要是飞燕草素-3-O-半乳糖苷含量比较高，而绿色品种中主要是锦葵色素-3-O-半乳糖苷含量比较高，而绿色品种中叶绿素a,b和总叶绿素含量明显高于红色和黄色品种。通过转基因研究了部分基因在番茄中的功能，相关基因的过量表达可以影响果实的色泽。对叶绿素含量，类胡萝卜素含量进行了测定，结果表明相关过量基因的表达可以影响上述物质的含量。